Trichomonas vaginalis is the most prevalent sexually-transmitted infectious organism in the United States with roughly 7 million people affected. Individuals infected with T. vaginalis face an increased risk of cervical cancer, infertility, pelvc inflammatory disease, prostatitis, and susceptibility to HIV. The nitroimidazoles are the only class of drugs available to treat trichomoniasis, but they suffer from several serious side effects Additionally, the emergence and spread of resistance to nitroimidazoles is becoming a concern. Consequently, there is a critical need for the development of new therapeutic agents to combat T. vaginalis and the proposed studies, designed in response to this RFA, provide a mechanism for the discovery and development of bioactive molecules that meet this need. Vital to the success of a drug discovery program is access to an outstanding library of chemically diverse substances that are well suited to become early stage leads. Natural products meet this essential requirement with metabolites from fungi having not been previously screened against T. vaginalis. Thus, this work is innovative since we will couple a novel natural product source with a new high-content screening system to identify promising bioactive substances from a large fungal secondary metabolite library. During the R21 phase of this investigation, we will focus on the following objectives to secure new compounds for treating T. vaginalis infections: (i) implement a high-content screening system for the detection of natural products that inhibit T. vaginalis viability; (ii) test a one-of-a-kind library of pure natural products, pre-fractionated materials, and secondary-metabolite-enriched fungal extracts for inhibitors of T. vaginalis; (iii) employ a mammalian-cell-based counter-screen to remove non-selective toxins from screening; and (iv) use fungal taxonomic information and secondary metabolite dereplication tools to select extracts and fractions for further studies. The most promising agents emerging from the R21 studies will be subjected to detailed R33 studies via the following aims: (i) purify bioactive natural products from fungal fractions and extract mixtures, (ii) establish the structures of bioactive metabolites and secure milligram quantities for biological evaluation, (iii) develop a complimentary high-content screening assay to evaluate each hit molecule against additional anaerobic protozoan targets (Giardia intestinalis and Entamoeba histolytica), and (iv) conduct in vitro metabolism studies of the most promising hit molecules and test their metabolites for toxicity and bioactivity. The successful accomplishment of these aims is expected to yield a focused set of natural products that inhibit T. vaginalis viability with minimal toxicity to human cells. It is anticipated that 3-5 promising inhibitors will be obtained that will serve as bioactiv leads for immediate investigation during subsequent R01/SBIR studies. The focus on securing high-quality bioactive compounds during the R21/R33 study is anticipated to help generate first-in-class molecules with applications for treating T. vaginalis and other anaerobic protozoans.